Mapping Mutations in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 14

VI. SNP Mapping

Assuming that only a single gene mutation is involved, the genotype of the F1s
derived from crossing a New mutant stock with CB4856 is new-1(*)/new-1(+),
where the new-1(+) allele is present on a CB4856-derived chromosome. Since
each F2 contains two independently derived recombinant genomes, for a section
of the genome that is unlinked to new-1 a given individual has equal likelihood
(25%) of being homozygous for either N2 sequence or CB4856 sequence, and
50% likelihood of being heterozygous. However, at the new-1 locus, individuals
that are homozygous for new-1(*) will have a 100% likelihood of being homo-
zygous for N2 sequence (and 0% chance of being heterozygous). As the distance
between a particular sequence and the new-1 locus increases, the likelihood of
SNP heterozygosity increases (up to a maximum of 50% at 50 cM). Since C.
elegans chromosomes are only 50 cM in length, and the majority of the genes on
each chromosome are located within the central gene clusters, assignment to
linkage group can usually be accomplished by testing only five centrally situated
SNPs, as described below (for recessive mutations, X-linkage can usually be
determined by inspecting the F1 progeny; dominant X-linked mutations are
distinguishable since F1 mutant males will transmit to 100% of their hermaph-
rodite progeny, but none of their male progeny. If the mutation prevents males
from mating, methods comparable to those used for autosomal mapping must be
used).

A. Bulked Lysate Analysis
Currently, the easiest method for SNP mapping is bulked lysate analysis
( Wicks et al., 2001 ). Here, I describe the use of ARMS-PCR (annealing restricted
marker system; Ye et al., 2001 ) for bulked lysate SNP analysis. This method uses
sequence-specific primer annealing to permit the assessment of SNPs that do not
alter restriction enzyme cleavage sites. Note, that by the time this chapter is
published technical advances and cost reductions might permit WGS to be eco-
nomically substituted for SNP mapping at

this stage of the analysis

( Doitsidou et al., 2010 ).

1. Set up two experimental lysates, one with New worms and the other with non-
New worms. Also set up three control lysates, one with only N2 worms, one
with only CB4856 worms, and one with equal numbers of CB4856 and N2
worms. To make a lysate, pick 100 animals into a PCR tube containing 200
m L of lysis buffer (50 mM KCL, 10 mM Tris pH 8.3, 2.5 mM MgCl 2 ,0.45%
NP-40, 0.45% Tween-20, 0.01% gelatin; keep stock frozen at 20 ,thawand
add proteinase K to 100 m g/mL immediately prior to use ( Williams et al.,
1992 )). Transfer the tube to 70 . After incubating at 70 for at least
10 min, transfer the tube to a PCR machine set to 60 for 4 h, followed by
95 for 15 min.